Equine herpesvirus type 1 (EHV-1) infection of the mouse offers a model to delineate molecular mechanisms by which a respiratory virus can elicit immunopathological responses in the lung, gain entry into the CNS, and mediate neurovirulence. Expression of a very few viral virulence determinants in EHV-1 respiratory infection shifts the host response from protection to severe inflammatory disease, an immunopathologic paradox similar to that of respiratory syncytiai virus infection in humans. Our recent data reveal that EHV-1 virulence is greatly influenced by the expression of viral glycoproteins gl and gE encoded within the Us genomic segment. Using the attenuated KyA and highly pathogenic RacL11 strains of EHV-1, we have generated recombinant EHV-1 (rEHV-1) differing in expression of gl and gE. Insertion of both gl and gE into KyA (Kgl/gE) restored the virulence phenotype to this attenuated EHV-1 and facilitated its entry and spread in the CNS. The deletion of gl and gE from pathogenic RacL11 did not result in full attenuation, suggesting RacL11 harbors an additional virulence determinant(s) not present in KyA. Our hypothesis is that this RacL11 virulence determinant is gp2, a large glycoprotein unique to EHV-1, that is encoded by Us gene EUs4. Expression of the full-size gp2 of 797 amino acids has been implicated in EHV-1 virulence. KyA harbors a major deletion of 414 residues within the potentially important highly variable domain of gp2. Our long-term objectives are to assess the importance of gl, gE, and variable forms of gp2 in EHV-1 respiratory pathogenesis and neurovirulence and to correlate their expression with the induction of cytokines/chemokines and cellular genes important in the immunopathologic outcome. In Aim 1, we shall construct rEHV-1 expressing various combinations of gl, gE, and gp2. The EUs4 gene of pathogenic RacL11 will be cloned and sequenced and used to generate rEHV-1 that express gp2 with different forms of its highly variable domain. In Aim 2, these rEHV-1 will be assessed in the CBA mouse for virulence, the ability to elicit pro- and anti-inflammatory cytokines/chemokines, and the capacity to enter and spread within the CNS. Cell types that infiltrate the lung and CNS and produce mediators of inflammation will be identified. In Aim 3, cell cultures will identify the cell types (e.g. Mo, T, B, and endothelial cells) that produce the mediators of inflammation following attachment and/or infection with various rEHV-1 known virulence phenotypes. These cell cultures as well as cells from the mouse will be used in our ongoing studies that employ DNA micro-array analyses to identify cell genes whose expression is differentially affected by infection with pathogenic versus attenuated EHV-1. Overall, completion of these aims will reveal how the expression of gl, gE, and/or various forms of gp2 influences EHV-1 virulence and spread, identify the target cell types and their cytokine/chemokine products that mediate severe inflammatory disease in the lung and CNS, and correlate alterations in the expression of cell genes with the virulence phenotype.